1. Field of the Invention
The present invention relates generally to the fields of cell culture and virus production. More particularly, it concerns improved methods for the culturing of mammalian cells, infection of those cells with adenovirus and the production of infectious adenovirus particles therefrom.
2. Description of Related Art
A variety of cancer and genetic diseases currently are being addressed by gene therapy. Viruses are highly efficient at nucleic acid delivery to specific cell types, while often avoiding detection by the infected host's immune system. These features make certain viruses attractive candidates as gene-delivery vehicles for use in gene therapies (Robbins and Ghivizzani, 1998; Cristiano et al., 1998). Modified adenoviruses that are replication incompetent and therefore non-pathogenic are being used as vehicles to deliver therapeutic genes for a number of metabolic and oncologic disorders. These adenoviral vectors may be particularly suitable for disorders such as cancer that would best be treated by transient therapeutic gene expression since the DNA is not integrated into the host genome and the transgene expression is limited. Adenoviral vector may also be of significant benefit in gene replacement therapies, wherein a genetic or metabolic defect or deficiency is remedied by providing for expression of a replacement gene encoding a product that remedies the defect or deficiency.
Adenoviruses can be modified to efficiently deliver a therapeutic or reporter transgene to a variety of cell types. Recombinant adenoviruses types 2 and 5 (Ad2 and AdV5, respectively), which cause respiratory disease in humans, are among those currently being developed for gene therapy. Both Ad2 and AdV5 belong to a subclass of adenovirus that is not associated with human malignancies. Recently, the hybrid adenoviral vector AdV5/F35 has been developed and proven of great interest in gene therapies and related studies (Yotnda et al., 2001).
Recombinant adenoviruses are capable of providing extremely high levels of transgene delivery. The efficacy of this system in delivering a therapeutic transgene in vivo that complements a genetic imbalance has been demonstrated in animal models of various disorders (Watanabe, 1986; Tanzawa et al., 1980; Golasten et al., 1983; Ishibashi et al, 1993; and S. Ishibashi et al., 1994). Indeed, a recombinant replication defective adenovirus encoding a cDNA for the cystic fibrosis transmembrane regulator (CFTR) has been approved for use in at least two human CF clinical trials (Wilson, 1993). Hurwitz, et al., (1999) have shown the therapeutic effectiveness of adenoviral mediated gene therapy in a murine model of cancer (retinoblastoma).
As the clinical trials progress, the demand for clinical grade adenoviral vectors is increasing dramatically. The projected annual demand for a 300 patient clinical trial could reach approximately 6×1014 PFU.
Traditionally, adenoviruses are produced in commercially available tissue culture flasks or “cellfactories.” Adenoviral vector production has generally been performed in culture devices that supply culture surfaces for attachment of the HEK293 cells, such as 1-flasks. Virus infected cells are harvested and freeze-thawed to release the viruses from the cells in the form of crude cell lysate. The produced crude cell lysate (CCL) is then purified by double CsC1 gradient ultracentrifugation. The typically reported virus yield from 100 single tray cellfactories is about 6×1012 PFU. Clearly, it becomes unfeasible to produce the required amount of virus using this traditional process. New scaleable and validatable production and purification processes have to be developed to meet the increasing demand.
The purification throughput of CsC1 gradient ultracentrifugation is so limited that it cannot meet the demand for adenoviral vectors for gene therapy applications. Therefore, in order to achieve large scale adenoviral vector production, purification methods other than CsC1 gradient ultracentrifugation have to be developed. Reports on the chromatographic purification of viruses are very limited, despite the wide application of chromatography for the purification of recbinant proteins. Size exclusion, ion exchange and affinity chromatography have been evaluated for the purification of retroviruses, tick-borne encephalitis virus, and plant viruses with varying degrees of success (Crooks, et al., 1990; Aboud, et al., 1982; McGrath et al., 1978, Smith and Lee, 1978; O'Neil and Balkovic, 1993). Even less research has been done on the chromatographic purification of adenovirus. This lack of research activity may be partially attributable to the existence of the effective, albeit non-scalable, CsC1 gradient ultracentrifugation purification method for adenoviruses.
Recently, Huyghe et al. (1996) reported adenoviral vector purification using ion exchange chromatography in conjunction with metal chelate affinity chromatography. Virus purity similar to that from CsC1 gradient ultracentrifugation was reported. Unfortunately, only 23% of virus was recovered after the double column purification process. Process factors that contribute to this low virus recovery are the freeze/thaw step utilized by the authors to lyse cells in order to release the virus from the c ells and the two column purification procedure. Of interest to the present invention is the disclosure of co-owned U.S. Published Patent Application No. 2004/0106184 A1, the disclosure of which is hereby incorporated by reference which is directed to methods for passing adenovirus particle preparations through chromatographic media to provide purified adenovirus particles.
For most of the E1 deleted first generation adenoviral vectors, production is carried out using HEK293 (human embryonal kidney cells, Invitrogen Corp.) cells which complement the adenoviral vector E1 deletion in trans. Because of the anchorage dependency of the HEK293 cells, adenoviral vector production has generally been performed in culture devices that supply culture surfaces for attachment of the HEK293 cells, such as T-flasks, multilayer Cellfactories™, and the large scale CellCube™ bioreactor system. Recently, the 11EK293 cells have been adapted to suspension culture in a variety of serum free media allowing production of adenoviral vectors in suspension bioreactors. Complete medium exchange at the time of virus infection using centrifugation is difficult to perform on a large scale. In addition, the shear stress associated with medium recirculation required for external filtration devices is likely to have a detrimental effect on host cells in a protein-free medium.
Of interest to the present invention are the disclosures of co-owned U.S. Pat. No. 6,194,191 and co-owned U.S. Pat. No. 6,726,907 the disclosures of which are hereby incorporated by reference, which are directed to improved Ad-p53 production methods with cells grown in serum-free conditions, and in particular in serum-free suspension culture. Also of interest to the present invention is the disclosure of WO 00/32754 based on U.S. Ser. No. 09/203,078, the disclosure of which is hereby incorporated by reference, which is directed to the use of low-medium perfusion rates in an attached cell culture system.
Clearly, there is a demand for improved methods of adenoviral vector production that will recover a high yield of product to meet the ever increasing demand for such products. Improved methods for adenoviral vector production can include improved techniques to make production more efficient, or optimization of operating conditions to increase adenoviral vector production.